This invention relates to flexible, autoclavable bioreactors and aquaculture systems incorporating said bioreactors.
A sterilizable, non-metallic bioreactor which is low cost, has utility in the production of autotrophic, mixotrophic and heterotrophic cell cultures and which can be incorporated into a wide range of bioprocessing systems. The bioreactor can be used, for example, for cell growth with subsequent extraction of cell components and also for applications that utilize the whole cell such as for the production of protein or for the production of algae for aquaculture.
In the pharmaceutical and food processing industries unicellular organisms are generally produced in commercial quantities using metallic bioreactors or using non-metallic systems that employ sterile plastic bags. Sterile plastic bag bioreactors may optionally be supported by an apparatus designed to rock the bag and mix its contents. In certain circumstances it is also possible to produce unicellular organisms in non-sterile bags.
Metallic systems are too expensive to be a viable method for commercial applications in agriculture and aquaculture. Rocking bag systems are also too costly for agriculture and aquaculture, and are limited in the extent to which they can be scaled up. Non-sterile bag systems cannot be used for mixotrophic (a heterotrophic reaction that is subject to photonic radiation) or heterotrophic cell culture because of the presence of bacteria that compete for the preferred food, soluble organic chemicals. Therefore, cell production utilizing non-sterile bags is restricted to autotrophic and photo autotrophic systems where significant quantities of soluble organic chemicals are not used as a source of nutrients. Photo autotrophic systems that employ artificial light as the energy source are inefficient and not cost effective and are therefore limited to commercial applications where the cost of cell production is not an important factor.
A practical, efficient bioreactor will find many applications in commercial microbiology and in new applications in agriculture and aquaculture. On-site production of unicellular food for animals is currently impractical because the commercial systems now in use for cell growth are too expensive for agriculture or aquaculture applications. The systems now in use are either glass-lined or stainless steel systems.
Rearing systems for zooplankton, shellfish and fish are well known and numerous versions of such systems are described in the literature. See, for example, U.S. Pat. No. 5,176,100. Rearing systems all have the same general requirements. One requirement is to reduce the concentrations of toxic metabolites in the water, for example ammonia. This requirement is generally met utilizing a biofilter through which the contaminated water is circulated and where the water is contacted with a stream of air. Nitrifying and other bacteria reside in the biofilter and serve to convert ammonia to less toxic nitrate and reduce the concentration of dissolved organic compounds. Alternatively, inorganic materials may be absorbed by zeolite or activated carbon, followed by contact with air or oxygen. Problems with the methods that employ absorption techniques include clogging of the filters and an insufficient contact of the unwanted materials with the oxygen.
A second requirement of a rearing system is the need for a high level of dissolved oxygen. This is commonly achieved by bubbling air or oxygen into the rearing tank water or through the use of an oxygen permeable bag systems as disclosed in U.S. Pat. No. 5,225,346.
A third requirement is the need to expeditiously remove solid waste materials because accumulations of solid waste can harbor colonies of toxic anaerobic bacteria. In existing systems solid wastes are generally removed through a connection in the bottom of the tank. The current systems often use grain-based nutrients. The problem with use of such systems is that grain-based foods result in solid wastes that contain lignin and highly oriented cellulose which are difficult to convert to algae nutrient or to any other practical use. Algae are the natural food for shellfish and zooplankton and, further, zooplankton is a natural food for fish and crustaceans. These natural foods create waste that may be catabolized by various enzymes that are common in nature, especially at the bottom of marine environments.
A fourth requirement of an efficient rearing system is the need to counter the build-up of nitrate in the rearing tank. In the past, this has usually been accomplished by purging the tank and thereafter discharging the purge into a waterway causing pollution.
One of the technological obstacles to the production of single-cell algae as a nutrient source is the need to separate the algae harvest from the culture. This obstacle is often overcome with techniques such as centrifugation, flocculation, reverse osmosis, etc. A continuous culture system integrated with rearing systems would provide significant efficiency benefits by eliminating the need to separate the cells from the media.
A variety of animal cell types can also be grown in culture, including connective tissue cells, skeletal, cardiac, and epithelial cells, neural cells, endocrine cells, melanocytes, and many types of tumor cells. Similarly a variety of growth media can be used, depending on the particular growth requirements of the cells and the growth conditions.
Animal cell culture systems can have a variety of configurations. Depending on the type of cells, their intended use, and the conditions of growth. Most cultures are propagated in the form of a monolayer, with the cells anchored to a glass or plastic substrate. Some, however, are preferably grown in suspension, which has the advantage of simpler propagation. Using a suspension system, subcultures can be made by simple dilution rather than by detaching (e.g., by trypsinization) the cells from anchored growth. Growth in suspension also provides increased surface area with increased bulk, as well as improved ease of harvesting, and the possibility of achieving a xe2x80x9csteady statexe2x80x9d culture.
Cell growth kinetics in suspension cultures can be affected by a number of considerations. Varying growth conditions such as the growth temperature, initial growth phase of cells, inoculation density, mixing rate, and medium surface area can have important effects. The selection of medium, nutrients, and pH all affect growth in a generally predictable, controllable manner. The ability to supply sufficient oxygen is particularly important and difficult in animal cell culture systems.
The present invention relates to an autoclavable, unicellular culture system comprising flexible, light transmitting walls, an internal oxygen diffuser, and a head plate which has a plurality of apertures.
The present invention further relates to a fish or shellfish rearing system incorporating the culture system.